【作者】 苗贵东；

【导师】 樊廷俊； 陈松林；

【作者基本信息】 中国海洋大学 ， 细胞生物学， 2010， 博士

【摘要】 半滑舌鳎为我国的本地种,主要分布于我国北方沿海。目前,半滑舌鳎的野生资源量非常稀少,渔业资源已处于非常严峻的形势,因此对遗传育种工作提出更高的要求。同时由于半滑舌鳎良好的经济价值,已与大菱鲆、牙鲆、条斑星鲽、圆斑星鲽一起成为北方重要的海水养殖鱼类。本研究从分子育种的实际需要出发,主要探讨以下四个关键问题,一是开发多态的微卫星标记；二是运用微卫星标记建立进行辅助家系管理的微卫星标记体系；三是多重PCR技术在半滑舌鳎亲子鉴定中的应用；四是构建基于微卫星标记的遗传连锁图谱。主要的研究结果如下：1.多态微卫星标记开发：采用用FIASCO方法构建了五个SSR富集文库,其核心序列分别为(GATA)6、(AAAC) 6、(ACTC)6、(ATC)8、(GA)13,从五个文库中分别挑取1000个单克隆进行PCR鉴定,有700个是阳性克隆,阳性率70%。随后,挑取插入片断大小在250bp-800bp之间的500个克隆进行测序,测序结果获得412条微卫星序列,阳性序列比例为82.4%,对这412个序列进行筛选、网上BLAST比对后,成功设计了273对引物。最终获得111个多态的微卫星位点。为半滑舌鳎的种质资源评估、家系选育、微卫星辅助家系管理、微卫星多重PCR体系建立、遗传连锁图谱构建等研究提供了候选标记。2.微卫星标记在半滑舌鳎系谱认证和家系管理中的应用采用本实验室开发和已发表的微卫星标记建立一个由10个微卫星组成的系谱认证微卫星体系,探讨对建立的Fl代16个家系473尾鱼进行系谱认证和亲子鉴定分析(个体识别)的可行性,依据基因型频率,Cervus2.0分析5-10个位点组合累积排除率,5对引物组合和10对引物组合的累积排除率分别为96.45%和99.71%。10个位点组合的亲子分析最后的结果实际识别率为95.35%(451/473)。由此可见,本研究建立的10个微卫星位点组合能够解决在家系选育初期无法进行物理标记时的辅助家系管理,同时通过遗传多样性的评价也能够更好的辅助进行亲本选择。3.多重PCR在半滑舌鳎亲子鉴定中的应用本研究获得半滑舌鳎两个微卫星多重PCR体系,包含9个微卫星引物,两个多重PCR的9个引物在16个家系160个个体中进行亲子分析和系谱认证结果表明,9个引物的平均等位基因数和多态信息含量分别为6.333和0.6333,Cervus2.0软件分析表明双亲未知条件下的累计排除率为95.66%,双亲中知道其中一个的累计排除率为99.65%,对基因型转化成(0,1)矩阵运用NTsys软件聚类分析的结果表明其聚类准确率为93.13%,两个多重PCR对16个家系个体进行亲子分析(Parentage analysis),亲子鉴定准确率为90%,结果表明,两个多重PCR能有效的进行半滑舌鳎的亲子分析,能够有效的辅助进行选育过程中的家系管理。4.半滑舌鳎遗传连锁图谱的构建本研究共使用微卫星标记1562个,微卫星标记来源有：来源于半滑舌鳎引物1076对；来源于条斑星鲽引物83对；来源于圆斑星鲽引物78对,来源于大西洋鳙鲽引物182对；来源于牙鲆引物96对,来源于赛内加尔鳎和欧洲鳎引物47对。上述引物经筛选共获得分离SSR标记319个用于构建SSR标记遗传连锁图谱。同时使用通过AFLP技术经克隆获得的雌性特异标记引物两对,分别构建雌、雄两张遗传连锁图谱。雌、雄图谱共定位291个标记,雌雄图谱共有标记166个。雌性连锁图谱共定位237个SSR标记、两个性别特异分子标记,分布于23个连锁群中。LOD值≥3.0,雌性连锁图谱每个连锁群的标记数目分别从2-20个不等,雌性图谱平均每个连锁群标记数平均为10.4个。雌性连锁图谱连锁群长度在28.2cM～116.2cM之间,相邻标记间最大间隔为58.6cM,平均间隔(s)为6.98cM。图谱总长度(Goa)为1668.2cM,预期长度(Ge)(基因组的预期大小)为2082.9cM,雌性图谱的基因组覆盖率(Coa)为80.09%。雄性连锁图谱共定位218个SSR标记,分布于20个连锁群中。LOD值≥3.0,雄性连锁图谱每个连锁群的标记数目2-32个不等,雄性图谱平均每个连锁群的标记数目为10.9。雄性连锁图谱连锁群长度在15.4cM～128.4 cM,相邻标记间最大间隔为46.4cM,平均间隔(s)为7.21cM。图谱总长度(Goa)为1571.7cM,预期长度(Ge)(基因组的预期大小)为1935.0cM,雄性图谱的基因组覆盖率(Goa)为81.23%。该图谱为全部基于SSR标记的半滑舌鳎遗传连锁图谱,图谱首次定位了几个与基因相关标记,该图谱能够用于该物种不同群体比较分析、为QTL定位、分子标记辅助育种提供了平台且为进一步构建高密度的遗传图谱奠定了基础。更多还原

【Abstract】 Half-smooth tongue sole(Cynoglossus semilaevis) is an important cultured marine fish species in the northern China. Half-smooth tongue sole which is the native fish species of China, mainly distributed in the north China coast. At present the number of wild individuals of the fish species is little, and the fisheries resource is rather critical.In aquaculture, It has been the most popular fish species together with turbot (Scophthalmus maximus), olive flounder (Paralichthys olivaceus), Spotted halibut (Verasper variegatus), and barfin flounder(Verasper moseri) in the northern China. In order to benefit the molecular breeding of fish species, the four items were studied in this paper including.1.Isolation and characterization of polymorphic microsatellite markersFive repeat-enriched genomic libraries (the repeat units were GATA, GA, ATC, ACTC and AAAC, respectively) were constructed by FIASCO (fast isolation by AFLP of sequences containing repeats) in Cynoglossus semilaevis.500 sequences were obtained, of which 412 contained microsatellite repeats.273 pairs of primers were designed successfully, and 111 microsatellite loci were proved to be polymorphic. These markers will be benefit for the evaluation of genetic resource and the construction of genetic linkage map in half-smooth tongue sole.2.Parentage determination of Cynoglossus semilaevis based on microsatellite DNA markers and its application to family management.This study was initiated to assess the feasibility of parentage determination in aquaculture populations for Cynoglossus semilaevsis.Under different scenarios both simulation and reality, we used microsatellite markers to assign parentage to offspring. Simulations based on allele frequency data from the population of Cynoglossus semilaevis demonstrated when marker data from five loci to ten loci were combined, the assignment success of progeny to their true parental couple from 96.45% increased to 99.71% in parentage identification with unknown parental and filial information. Out of 473 offspring,95.35%(451/473)was assigned to their true parental couple in mixed families groups. Based on these results, we conclude that the use of a number of microsatellite markers represents a realistic and effective alternative to physical tagging in a selection program and it allows the identification of parental effects on offspring performances from early life stages.3.Development of two microsatellite multiplex PCR systems for Cynoglossus semilaevis and its application to family management.We developed two microsatellite multiplex PCR systems,9 microsatellite loci,for Cynoglossus semilaevis, one of the most important aquaculture species in China. The multiplex PCR microsatellite assay was used to estimate the kinship and the genetic diversity of sixteen Cynoglossus semilaevis families.The average polymorphic information content (PIC) was 0.6333.The number of the alleles at each locus ranged from three to eight.The number of the average alleles was 6.333.Based on the genotypes of either known parent or none of them and offspring all the absent parental genotypes of the sixteen families were deduced.According to the unique alleles among the families,each family could be distinguished from the others.Cervus2.0 analysis showed that the combined exclusion probability was 95.66% if parents were both unknown and the combined exclusion probability was 99.65% when only one of the parental genotype was known. A dendrogram was constructed using UPGMA based on genetic similarity matrix.The UPGAM dendrogram of the 160 individuals indicated that 93.13% the individuals from each family could be fully clustered and the classification results accord with the source of genealogy.In fact 90% correct assignment was performed with two microsatellite multiplex PCR. The results showed that microsatellite marker is useful tool for general logical identification of Hat-Smooth Tongue Sole.4. The construction of genetic linkage mapsTwo genetic linkage maps of Cynoglossus semilaevis.(♀) and Cynoglossus semilaevis (♂) were constructed using microsatellite markers. A total of 289 SSR markers and 2 female-specific AFLP markers were mapped in genetic linkage maps of Cynoglossus semilaevis.The genetic linkage maps of Cynoglossus semilaevis (♀) map consisted of 237 SSR markers and 2 female-specific AFLP markers, and it was grouped in 23 linkage groups.The linkage between the loci was identified by LOD score of≥3.0. The number of markers per group ranged from 2 to 20, with an average of 10.4. The length of groups ranged from 28.2cM-116.2cM (Kosambi), with an average distance of 6.98cM between markers. The observed consensus map length and the estimated consensus map length were 1668.2cM and 2082.9cM, respectively, with the coverage of 80.09%.The genetic linkage maps of Cynoglossus semilaevis (♂) consisted of 218 SSR markers and it grouped 20 linkage groups.The number of markers per group ranged from 2 to 32,with an average of 10.9.With an average resolution of 7.21 cM.The observed consensus map length and the estimated consensus map length were 1571.7cM and 1935.0cM, respectively, with the coverage of 81.23%.186 microsatellite markers were common to both maps. It is the first time to construct SSR genetic linkage maps for Cynoglossus semilaevis which could be useful for quantitative trait loci (QTL) mapping, MAS (molecular marker-assisted breeding) and construction of high density linkage map of this species fish.更多还原